Studies on the biology of black mould (Aspergillus niger) on temperate and tropical onions. 1. A comparison of sources of the disease in temperate and tropical field crops

Aspergillus niger colonies were present on 60–90% of the leaves sampled from onion crops grown under hot, tropical conditions (Shambat, Sudan), but only on 21% of leaves in crops grown under temperate conditions (Wellesbourne, UK). The seed stocks used in the Sudan were naturally contaminated with A. niger , whereas the fungus was absent from seed used in the UK. Inoculation of seed with A. niger resulted in an increase in the incidence of the fungus on onion plants (mean 83% of plants with affected leaves) in the field and on bulbs in store in crops grown in the UK. In the UK, A. niger is infrequent in the soil and air, but it is a major component of the soil and air mycoflora in the Sudan. Most of the bulbs grown in Sudan were contaminated with A. niger at harvest because of the high incidence of the fungus in the field.     

Sources, survival and transmission of Cladosporium allii and C. allii-cepae, leaf blotch pathogens of leek and onion

Neither Cladosporium allii nor C . allii-cepae was detected in seed samples of 46 commercial cultivars of leek and nine of onion.
In glasshouse tests where flower inflorescences of onions and leeks were dusted with conidia of C. alliicepae and C. allii , respectively, few onion seeds and no leek seeds became contaminated. In field crops of leeks some spathes and inflorescences became diseased and gave 0 002% C. allii contaminated seeds. No infected onion seeds were obtained from naturally-infected plants in the field.
Conidia of both fungi survived for less than 6 weeks in sterile and non-sterile soil (during which time the temperature varied from -8 to +11 C); pseudothecial bodies survived longer.
Both pathogens survived for 3 months in debris of onion or leek on the surface of soil in pots under similar conditions. In debris buried at a depth of 7 cm in sterile or unsterile soil they survived for 2 months. Although C. allii survived only 1 month in debris on the soil surfaces following a leek crop, conidia of the fungus were trapped from the air above the area for at least 6 months and healthy leeks transplanted into this area developed the disease. No leaf blotch developed in leeks grown on land which had not previously borne that crop.     

Tracing seed to seedling transmission of the wheat blast pathogen Magnaporthe oryzae pathotype Triticum

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT), initially restricted to South America, is a global threat for wheat after spreading to Asia in 2016 by the introduction of contaminated seeds, raising the question about transmission of the pathogen from seeds to seedlings, a process so far not well understood. We therefore studied the relationship between seed infection and disease symptoms on seedlings and adult plants. To accomplish this objective, we inoculated spikes of wheat cv. Apogee with a transgenic isolate (MoT-DsRed, with the addition of being resistant to hygromycin). We identified MoT-DsRed in experiments using hygromycin resistance for selection or by observation of DsRed fluorescence. The seeds from infected plants looked either apparently healthy or shrivelled. To evaluate the transmission, two experimental designs were chosen (blotter test and greenhouse) and MoT-DsRed was recovered from both. This revealed that MoT is able to colonize wheat seedlings from infected seeds under the ground. The favourable conditions of temperature and humidity allowed a high recovery rate of MoT from wheat shoots when grown in artificial media. Around 42 days after germination of infected seeds, MoT-DsRed could not be reisolated, indicating that fungal progression, at this time point, did not proceed systemically/endophytically. We hypothesize that spike infection might occur via spore dispersal from infected leaves rather than within the plant. Because MoT-DsRed was not only successfully reisolated from seed coats and germinating seeds with symptoms, but also from apparently healthy seeds, urgent attention is needed to minimize the risks of inadvertent dispersal of inoculum.     

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.     

Detection of Diplodia sapinea in Corsican pine seeds

Diplodia sapinea is one of the major pathogens of pines worldwide. Despite the putative critical importance of seed infection in the epidemiology of the disease, this aspect of the biology of the fungus is poorly known. Here, biological and molecular methods were developed for the detection of the fungus and applied to assess D. sapinea infection in Corsican pine seeds. A buffered medium containing tannic acid and malt extract as a nutrient base was the most efficient and selective for D. sapinea recovery. A molecular method based on DNA extraction with a commercial kit and specific amplification, including an internal amplification control, was developed. A high percentage of infection (57% positive isolations) was observed in seeds obtained from fallen cones in a Corsican pine stand with no apparent symptoms of D. sapinea. Seeds collected from trees in a seed orchard showing severe symptoms of dieback caused by D. sapinea had comparatively lower infection (38%). Moreover, very low infection levels (1–5%) were observed after the standard treatment used for seed extraction, which included heating at 40°C. Diplodia sapinea was not recovered from seedlings grown from infected seed lots submitted to water stress. Overall, results suggest that the risk of disease transmission by commercial seeds is probably low, but could be further reduced by thermotherapy.     

Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

棉花黄萎病种子内部带菌的研究

1964—1966三年共分离采自严重黄萎病株下部的尚未开裂成铃种子11505粒,平均内部带菌率<0.026%。系统分离棉铃各部位,其带菌率的趋势为:铃柄>铃托>胎座。从已吐絮的成熟铃内种子内部带菌分析结果看:1964年共分离外部带菌率约为5%的种子1557粒,全部未长微菌核。1966年分离病铃内成熟种子4049粒,只有一粒在种壳上长出了微菌核,带菌率为0.025%。用浓胞子液在田间灌注接种初裂铃,依受病程度不同,种子内部带菌率为0.93—2.4%。青铃注射接种的内部带菌率为10.40—70.40%。新吐絮铃喷洒接种,虽保湿1—2天,种子内部未分离到病菌。说明在菌量大,营养及温、湿度条件非常适宜时,病菌也可自种壳侵入内部,使有相当高的带菌率。通过三年的试验,作者肯定在自然情况下黄萎病菌主要附着在棉籽短绒上。内部带菌率极低。在轻病或无病棉株上选收正常吐絮铃内种子,内部基本不带菌。     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the detection of loose smut of barley (Ustilago nuda)

Polyclonal antibodies were raised against mycelium from the logarithmic growth phase of a shake culture of Ustilago nuda, and a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) with biotinylated detection antibodies was developed. The detection limit of the assay was 15 ng total protein ml^–1 for the homologous antigen and 50 ng ml^–1 for a spore extract, respectively. Other species of Ustilago reacted with the antibodies. Cross-reactivity was highest with U. tritici. No signal was obtained with the tested isolates of Tilletia, Rhizoctonia, Pythium and Fusarium. With naturally infected barley seeds, the results of the ELISAs were always in good agreement with those obtained with the routinely used seed embryo test. However, when seeds grown from artificially inoculated florets were used, the ELISA indicated significantly higher infestation levels than the embryo test. Results of assays with halved seeds from the same lot showed that high amounts of mycelium were present in the non-embryo half. This and especially the relatively long duration of the assay suggested that the ELISA (as conducted here) may not be suitable as a routine method for analysing seed infection with U. nuda. With samples from barley seedlings grown from infected seeds the results of the immunoassay again corresponded very well with the infection level determined by staining of the seed embryo, irrespective of the mode of floret inoculation (natural or artificial). Potential fields of application of the ELISA include the early prediction of the efficacy of protection agents, e.g. in screenings for seed treatments, the elucidation of the biology of the fungus and characterisation of resistance mechanisms.     

Airborne spores of Tilletia caries as a source of wheat bunt through soil contamination

In 1985 severe bunt was encountered in a crop of wheat grown from organomercury-treated seed. Circumstantial evidence suggested that the soil had been contaminated by airborne spores of the fungus from a nearby crop which was being harvested at the same time as the affected crop was being sown.     

Presence of Xylella fastidiosa in Sweet Orange Fruit and Seeds and Its Transmission to Seedlings

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium, causes several economically important diseases in North, Central, and South America. These diseases are transmitted by sharpshooter insects, contaminated budwood, and natural root-grafts. X. fastidiosa extensively colonizes the xylem vessels of susceptible plants. Citrus fruit have a well-developed vascular system, which is continuous with the vascular system of the plant. Citrus seeds develop very prominent vascular bundles, which are attached through ovular and seed bundles to the xylem system of the fruit. Sweet orange (Citrus sinensis) fruit of cvs. Pera, Natal, and Valencia with characteristic symptoms of citrus variegated chlorosis disease were collected for analysis. X. fastidiosa was detected by polymerase chain reaction (PCR) in all main fruit vascular bundles, as well as in the seed and in dissected seed parts. No visual abnormalities were observed in seeds infected with the bacterium. However, the embryos of the infected seeds weighed 25% less than those of healthy seeds, and their germination rate was lower than uninfected seeds. There were about 2,500 cells of X. fastidiosa per infected seed of sweet orange, as quantified using real-time PCR techniques. The identification of X. fastidiosa in the infected seeds was confirmed by cloning and sequencing the specific amplification product, obtained by standard PCR with specific primers. X. fastidiosa was also detected in and recovered from seedlings by isolation in vitro. Our results show that X. fastidiosa can infect and colonize fruit tissues including the seed. We also have shown that X. fastidiosa can be transmitted from seeds to seedlings of sweet orange. To our knowledge, this is the first report of the presence of X. fastidiosa in seeds and its transmission to seedlings.     

Seed transmission of<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">lactucae</Emphasis>

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed. http://www.phytoparasitica.org posting Dec. 16, 2003.     

The dormancy of wild oat seed (Avena fatua L.) from plants grown under various temperature and soil moisture conditions

Wild oat plants of types fA, fB and fC were grown at a constant 15 or 20°C during the period of seed maturation. Seed of the three types differed little in dormancy when grown at 15°C, but at 20°C a larger proportion of seeds of type fA were dormant compared with fB or fC. Overall, dormancy of seed produced at 15 and 20°C was 97 and 63% respectively. Plants of another collection of type fB were grown from seed at 15 or 20°C with or without water stress applied only from the time of panicle emergence. Water stress and high temperature reduced viable seed production. Seed dormancy was tested immediately after collection by planting the seed in soil, and by Petri dish tests. Further Petri dish tests were made after 6 months storage. Seedling emergence in the first autumn from seeds of plants matured without water stress at 15°C was 10% compared with 30% for seeds grown at 20°C. Seeds grown with water stress at 15°C gave 47%, and at 20°C 78% emergence. The majority of emergence from seeds formed at 15°C without water stress occurred in the second spring after burial. Petri dish tests support these findings and suggest that seeds produced in hot dry summers are less dormant than those produced in cool moist ones.     

Isolation of Microdochium oryzae and Pinatubo oryzae from Rice Seeds and their Survival on Stored Seeds

The aim of these studies was to develop a semi-selective medium to differentiate Microdochium oryzae and Pinatubo oryzae, determine the frequency of seed infection of M. oryzae, study survival of the pathogen in stored seeds, and determine the frequency of infection of seed components. To simulate epidemics of differing intensities, panicles of rice cultivars that are susceptible (IR36) and resistant (IR42 and IR46) to M. oryzae were either non-inoculated, inoculated once, twice, or three times with a conidial suspension of M. oryzae. Both M. oryzae and P. oryzae colonies were recovered from seeds and were similar in culture. A semi-selective medium developed to detect M. oryzae seed infection rates aided in differentiating M. oryzae and P. oryzae by stimulating aerial conidiogenesis of P. oryzae. The conclusions taken from these results were: (a) seeds of IR36 had higher infection of M. oryzae than of IR42 and IR46 from plants grown in the dry season, but had lower infection of M. oryzae than of IR42 and IR46 from plants grown in the wet season; (b) M. oryzae infected seeds increased with an increase in the epidemic intensity with the highest occurring after three inoculations, the least occurring with non-inoculation, and intermediate with one, or two inoculations; (c) survival of M. oryzae decreased over time in seed lots stored at 10°C and 40% relative humidity and (d) all components of the rice seeds of IR36, IR42 and IR46 lots were infected with M. oryzae with the highest frequency in the endosperm and lemma, intermediate in the basal glumes and palea, and the least in the embryo.     

Studies on the biology of black mould (Aspergillus niger) on temperate and tropical onions. 2. The effect of treatments on the control of seedborne A. niger

Treatment of A. niger -inoculated onion seed with benomyl dust (1 g a.i./kg), or a foliar spray of thiram (0·4% a.i./ha) applied to plants grown from inoculated seed under temperate (UK) conditions reduced the incidence of A. niger in harvested crops. Treatment of naturally contaminated Sudanese onion seed with a benomyl + thiram mixture at a rate of 2·5 + 2·5 g a.i./kg or soaking the seed in hot water (15 min at 60°C) reduced the incidence of black mould on bulbs grown in the Sudan in field soil that had not previously been used for onion production. The seed treatments were less effective in crops produced in fields regularly used for onion production. However, incubation of harvested bulbs in moist chambers showed that damage to the internal storage tissue of onion bulbs caused by A. niger was reduced by seed treatment.     

Detection of <Emphasis Type="Italic">Phoma valerianellae</Emphasis> in lamb’s lettuce seeds

After the reappearance in Italy of a foliar disease of lamb’s lettuce (Valerianella olitoria L. Poll. syn. V. locusta L. Betcke) incited by Phoma valerianellae, we set out to measure the level of seed infection by this fungus, using a plating test, and to develop a molecular method for quick and reliable detection of the pathogen in seeds. All six samples of lamb’s lettuce seed tested were contaminated by P. valerianellae at levels of 0.6% to 15%. Surface disinfection of seeds did not eliminate the contamination and only reduced it to between 0.1% and 10%. The need for a sensitive, reliable and rapid diagnostic method for early identification of the fungus exists. We have developed a PCR-based method to identify the fungus in seeds. Variation within the internal transcribed spacer (ITS1, 5.8S sequences and ITS2) region of the rDNA (ITS) was used to characterize the P. valerianellae strains and to design specific primers within the ITS region.     

Biology of Commelina benghalensis L. in south-eastern Queensland. 1. Growth, development and seed production

Experiments conducted at Kingaroy in southeastern Queensland, investigated growth, development and seed production of Commelina benghalensis L. a weed of field crops, which produces aerial and underground flowers. Plants were grown from four seed types (large and small seeds from the two types of flowers) in pots exposed to the weather and in the field. Development was faster, and growth and seed production were greater in the field than in pots. Plants grown from aerial seeds were smaller, developed aerial flowers earlier, and produced more aerial fruits than plants grown from underground seeds. Rhizomes and underground flowers began to develop 6 weeks after emergence on plants from all seed types, prior to the development of aerial flowers. Seed production in the field was 8000 m^?2 for plants grown from underground seeds and 12000 m^?2 for plants grown from aerial seeds. Small aerial seeds represented 73–79% of the total seed production, large aerial seeds 19–22%, and underground seeds only 1–3%.     

Studies on inoculum sources of angular leaf spot of beans caused by Phaeoisariopsis griseola in Uganda

Abstract

Angular leaf spot of beans caused by Phaeoisariopsis griseola is a major problem on this crop in Eastern Africa. The sources of inoculum for this disease were investigated. The causal fungus was confirmed as seedborne in all the cultivars tested. The fungus caused seed discolouration but not all infected seeds were discoloured. Seed to seedling transmission was low. The fungus survived in infected crop debris for a maximum of nine and four to six months under indoor and outside conditions respectively. Under soil, the fungus survived for only two months. Infected offseason crops and volunteer plants were present at the time of planting the seasons’ crops and were an obvious source of the inoculum. It is concluded that the seed, crop debris, off‐season crops and volunteer plants are all possible sources of P. griseola infection under the local conditions.     

Colonization of citrus seed coats by 'Candidatus Liberibacter asiaticus': implications for seed transmission of the bacterium

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.     

Virulent and fungicide-tolerant races of loose smut (Ustilago nuda and U. tritici) in Ireland

Following the severe outbreaks of loose smut in the early 1980s, a survey was carried out over the period l984-86of the Ustilago nuda and U. tritici populations in the Republic of Ireland. U. tritici was very rare, only one infected wheat plant being reported in the 3 years of the survey. This fungus was represented by a virulent race distinct from those identified in Britain in the 1950s. U. nuda was widespread, and two races were identified in 1984. A virulent race, capable of overcoming the recessive resistance gene present in differential variety Missouri Early Beardless, was isolated from a single crop grown from imported seed. Several crops grown from seed treated with Vitavax (active ingredient carboxin), which had been imported from France, contained a carboxin-tolerant race of U. nuda. Subsequent surveys in 1985 and 1986 failed to recover these U. nuda races. The role of imported infected seed in the introduction of new races of the loose smut fungi is discussed.